1. Field of the Invention
The present invention relates to a method for enhancing a polymerase activity as well as a composition and a reaction mixture used for said method, which are useful in the field of genetic engineering.
2. Description of Related Art
A polymerase is one of the most important enzymes in the field of genetic engineering. Various additives such as DMSO, glycerol, formamide and poly(ethylene glycol) have been reported to enhance a polymerase activity. A surfactant is also one of additives for enhancing a polymerase activity. It is known that a nonionic surfactant stabilizes a thermostable DNA polymerase (Japanese Patent No. 2719528 (corresponding to U.S. Pat. No. 6,127,155)). Furthermore, it has been reported that a cationic surfactant, polyethoxylated amine, stabilizes a polymerase (Japanese Patent No. 3673175 (corresponding to U.S. Pat. No. 6,242,235)). In this document, it is predicted that high affinity ionic bonds protect a protein from denaturation by other active substances, and it is shown that ionic surfactants stabilize a polymerase better than nonionic surfactants. Specifically, the charged groups on the ionic surfactant electrostatically interact with the charged residues on the surface of the protein, the hydrophobic region in the ionic surfactant hydrophobically binds to the hydrophobic site in the protein, and the protein is protected due to the noncovalent crosslinking action. However, it is feared that a cationic surfactant may interact with an anionic nucleic acid molecule as a substrate for a polymerase to reduce the reactivity of the polymerase, although it depends on the concentration of the added surfactant.
Anionic surfactants such as sodium dodecyl sulfate (SDS), sodium deoxycholate, sodium N-lauroyl sarcosinate are known to inhibit a polymerase activity (Weyant, R. S. et al., Bio Techniques, 1990, Vol. 9, p. 308-309). Such anionic surfactants inhibit a polymerase activity even at low concentrations (e.g., 0.01% of SDS). Thus, if a polymerase reaction mixture is contaminated with an anionic surfactant used in pretreatment such as a nucleic acid extraction step, the polymerase activity is considerably inhibited.
With the progress in genetic engineering techniques, large-scale reading of nucleotide sequences and replication of a large amount of nucleic acid are routinely conducted. The influence of enhancement of a polymerase activity is great even if the degree is low. Thus, an additive that enhances a polymerase activity more effectively than conventional additives has been desired. Furthermore, an additive having an effect that is not brought by conventional additives has also been required.